Aerobic organisms are subjected to oxidative stress from many sources, including atmospheric oxygen, basal metabolic activities, and, in the case of pathogenic microorganisms, toxic oxidants from the host phagocytic response intended to destroy the bacterial invader. Glutathione (GSH) is the dominant low molecular weight thiol in most eukaryotes and Gram-negative bacteria, and it plays a key role in protection of the cell against oxygen toxicity and electrophilic toxins (R. C. Fahey and A. R. Sundquist (1991) Adv. Enzymol. 64:1–53; Dolphin, et al, (1989) Glutathione: Chemical, Biochemical, and Medical Aspects pp 45–84, John Wiley & Sons, New York). However, actinomycetes, including Streptomyces and Mycobacteria do not make GSH but produce millimolar levels of mycothiol (MSH, AcCys-GlcN-Ins), an unusual conjugate of N-acetylcysteine (AcCys) with 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins) (G. L. Newton, et al. (1996) J. Bacteriol. 178:1990–1995; S. Sakuda, et al., (1994) Biosci. Biotech. Biochem. 58:1347–1348; H. S. C. Spies and D. J. Steenkamp, (1994) Eur. J. Biochem. 224:203–213; G. L. Newton, et al. (1995) Eur. J. Biochem. 230:821–825) (FIG. 1A).
Mycothiol autoxidizes more slowly than glutathione (G. L. Newton, et al. (1995) Eur. J. Biochem. 230:821–825) and mutants of Mycobacterium smegmatis defective in the biosynthesis of mycothiol have increased sensitivity to hydrogen peroxide and antibiotics relative to the parent strain (G. L. Newton, et al. (1999) Biochem. Biophys. Res. Commun. 255:239–244). This observation suggests that mycothiol may play a key role in the protection of actinomycetes against oxygen toxicity and reactive toxins. The biochemistry of mycothiol appears to have evolved completely independently of that of glutathione.
However, it has already been established that the metabolism of mycothiol parallels that of glutathione metabolism in two enzymatic processes. First, formaldehyde is detoxified in glutathione-producing organisms by NAD/glutathione-dependent formaldehyde dehydrogenase (L. Uotila, et al. (1989) in Glutathione: Chemical, Biochemical, and Medical Aspects—Part A (D. Dolphin, et al., Eds.) pp 517–551, John Wiley & Sons, et al.). An analogous process involving NAD/mycothiol-dependent formaldehyde dehydrogenase has been identified in the actinomycete Amycolatopsis methanolica (M. Misset-Smits, et al. (1997) FEBS Lett. 409:221–222). This enzyme has been sequenced (A. Norin, et al. (1997) Eur. J. Biochem. 248:282–289).
A mycothiol homolog of glutathione reductase was recently cloned from M tuberculosis and expressed in M. smegmatis (M. P. Patel, et al. (1999) J. Amer. Chem. Soc. 120:11538–11539, M. P. Patel, et al. (1999) Biochem. 38:11827–11833). The reductase is reasonably specific for the disulfide of mycothiol but is also active with the disulfide of AcCys-GlcN, the desmyo-inositol derivative of mycothiol (M. P. Patel, et al. (1999) supra.). Therefore, there is a need in the art for investigation of the details of the metabolism of mycothiol and comparison with the established roles for the metabolism of glutathione.
Antibiotic resistance of pathogenic bacteria, including pathogenic actinomycetes, such as M. tuberculosis, is a well-known problem faced by medical practitioners in treatment of bacterial diseases. Therefore, there is a further need in the art for screening techniques to discover new antibiotics and drugs effective to reduce resistance to existing antibiotics in treatment of bacterial infections in humans and in other mammals, such as domestic and farm animals.
Air, soil and groundwater in areas surrounding industrial centers and farming areas are becoming increasingly polluted with simple organic compounds with have long lifetimes in the environment. These compounds include, but are not limited to 1, 2 dibromoethane, 1,2 dichloroethane, perchloroethene, trichloroethene, isoprene, and vinyl chloride. They are from pesticides, industrial degreasers, solvents, and from the production polyvinyl chloride polymers (plastics). Organisms have recently been isolated from contaminated environments that have the ability to detoxify, and in some cases grow using these pollutants as a sole carbon source. There is great interest in industrialized countries in using microorganisms for biodegredation of these pollutants in soil and groundwater, a field generally known as bioremediation.
Recent reports indicate that vinyl chloride, 1,2 dibromoethane, and numerous other haloalkanes are detoxified by mycobacteria (A. Jesenke et al., Microbiology, 66:2219–222 (2000); S. Hartmans, and A. M. DeBont, Applied and Environmental Microbiology, 58:1220–1226 (1992).; G. J. Poelarends, et al. J. Bacteriol, 181:2050–2058, (1999)). These toxic compounds are generally dehalogenated to form epoxides or monohaloaldehydes that are in turn toxic compounds to microorganisms until they are conjugated with thiols. Many of these organisms are actinomycetes and are likely to have mycothiol and mycothiol biosynthesis (G. L. Newton, et al. (1996) J. Bacteriol., 178:1990–1995). In the case of mycobacteria, mycothiol is the major low molecular weight thiol and will form a mycothiol conjugate. The product of this conjugation may still be toxic. Although such studies have shown the need for low molecular weight thiols in the detoxification reactions for toxins and have assayed their organisms for glutathione, to date such studies have not acknowledged the occurrence of mycothiol in mycobacteria.
Another actinomycete, Rhodococcus sp. Strain AD45 has been extensively studied for detoxification of isoprene, 1,2 dibromoethane and 1,2 dichloroethene (J. E. T. van Hylckama Vlieg, et al., Current Opinion in Microbiology, 3:257–262 (2000)). The enzymes responsible for the detoxification of these toxic substances were claimed to include a glutathione S-transferase and a glutathione conjugate specific dehydrogenase (J. E. T. van Hylckama Vlieg, et al, Applied and Environmental Microbiology, 64:2800–2805 (1998); J. E. T. van Hylckama Vlieg, et al. J. Bacteriology, 181:2094–2101 (1999); J. E. T. Van Hylckama Vlieg et al., J. Bacteriology 182:1956–1963 (2000).
Thus, there is a further need in the art for methods and compounds useful for detoxification of environmental toxins.